Caspase-mediated degradation of human 5-lipoxygenase in B lymphocytic cells

Abstract

5-Lipoxygenase (5-LO) is a tightly regulated enzyme in the synthesis of bioactive lipids from arachidonic acid. Here, we demonstrate that 5-LO is regulated by caspases, which are signaling molecules that control critical biological processes by means of specific limited proteolysis. Cell splitting of the Epstein-Barr virus-transformed B lymphocytic cell line BL41-E95-A caused a pronounced, but transient, reduction of functional 5-LO protein, accompanied by the appearance of a 62-kDa 5-LO cleavage product. In parallel, splitting of BL41-E95-A cells induced activation of caspase-6 (casp-6) and casp-8. Caspase activation and 5-LO degradation were blocked by the protein-synthesis inhibitor cycloheximide, and cell-permeable peptide inhibitors of casp-6 and casp-8 prevented 5-LO cleavage. Activation of casp-6 and casp-8 was connected to subsequent enhancement of cell proliferation, whereas selective caspase inhibition blocked cell growth. Last, isolated human 5-LO was cleaved by recombinant casp-6 in vitro to a 58-kDa fragment. Based on site-directed mutagenesis studies, 5-LO is cleaved by casp-6 after Asp-170, which in a homology-based 3D model of 5-LO is located on the enzyme periphery. We suggest that splitting of BL41-E95-A cells induces de novo synthesis of a protein involved in the activation of casp-6, which cleaves 5-LO.

Fig. 1.

5-LO in BL41-E95-A cells is down-regulated by splitting. BL41-E95-A cells, grown to 1.5–2 × 10⁶ cells per ml within 4 days, were diluted into fresh medium to 0.2 × 10⁶ cells per ml and harvested after the indicated times. The data are representative of four independent experiments. Values are given as mean + SE (n = 4–6). (A) 5-LO activity was assayed as described in Materials and Methods and is expressed as percentage of the maximal value (100%) obtained during the time-course study (days 0–10). (B) 5-LO protein and β-actin (control) were analyzed by WB. Total cell lysates corresponding to 0.8 × 10⁶ cells (15–20 μg of protein) were applied. The molecular mass of full-length 5-LO (78 kDa) and the 5-LO fragment (62 kDa) are indicated. (C) RNA was isolated; cDNA was prepared; PCR was performed by using a primer for 5-LO and β-actin; and the amplification products were analyzed as described.

Fig. 2.

Transfer to fresh medium and dilution of the cell density is required for down-regulation of 5-LO. BL41-E95-A cells, grown to 1.5–2 × 10⁶ cells per ml within 4 days in regular growth medium, were left untreated (I), diluted into the same medium to 0.2 × 10⁶ cells per ml (II), transferred into fresh medium without dilution (III), or diluted into fresh medium to 0.2 × 10⁶ cells per ml (IV). After 36 h, cells were harvested and 5-LO activity was determined in homogenates. 5-LO and β-actin protein levels were assayed by WB of total cell lysates corresponding to 0.8 × 10⁶ cells. The data are representative of four independent experiments. Values are given as mean + SE (n = 4). **, P < 0.01, by Student's t test.

Fig. 3.

Effects of CHX on 5-LO activity and protein levels. BL41-E95-A cells, which were grown for 4 days to 1.5–2 × 10⁶ cells per ml, were split into fresh medium to 0.2 × 10⁶ cells per ml or left untreated. CHX (50 μM) was added as indicated, and cells were cultured for another 24 h. 5-LO activity in homogenates (graph) and 5-LO protein levels (blot) were assayed. Values are given as mean + SE (n = 4). **, P < 0.01, by Student's t test. Results of WB (total cell lysates corresponding to 0.8 × 10⁶ cells) are representative of four independent experiments.

Fig. 4.

Cell splitting induces activation of casp-6 and casp-8. Effects of CHX are shown. (A) BL41-E95-A cells, grown to 1.5–2 × 10⁶ cells per ml within 4 days, were split into fresh medium in the presence or absence of 50 μM CHX, or left untreated. After 24 h, the activities of casp-6 and casp-8, casp-8 processing, and 5-LO protein degradation were determined. For WB (also see B), total cell lysates corresponding to 0.8 × 10⁶ cells were applied. (B) Time course of casp-8 activity, processing of casp-6 and casp-8, and 5-LO protein degradation. BL41-E95-A cells, grown to 1.5–2 × 10⁶ cells per ml within 4 days, were split into fresh medium, harvested after the indicated periods, and analyzed as indicated. (C) BL41-E95-A cells, which were grown to 1.5–2 × 10⁶ cells per ml within 4 days, were split into fresh medium and the increase in cell numbers, and cell viability was assessed by light microscopy using trypan-blue exclusion. Values are given as mean + SE (n = 4).

Fig. 5.

Effects of caspase inhibitors on 5-LO cleavage and proliferation of BL41-E95-A cells. (A) BL41-E95-A cells, grown to 1.5–2 × 10⁶ cells per ml within 4 days, were split into fresh medium and grown for 48 h in the presence or absence of VEID-CHO (10 μM) and IETD-CHO (6 μM), respectively. 5-LO protein (total cell lysates corresponding to 0.8 × 10⁶ cells) was analyzed by WB. (B) BL41-E95-A cells, which were grown to 1.5–2 × 10⁶ cells per ml within 4 days, were split into fresh medium and grown for 48 h in the presence of the indicated concentrations of VEID-CHO. Cell numbers and viability were assessed by light microscopy using trypan-blue exclusion, and 5-LO protein was assessed by WB. Cell growth is given relative to the cell number at split. The results are representative of at least five experiments. Values are given as mean + SE (n = 4). **, P < 0.01, by Student's t test.

Fig. 6.

5-LO is cleaved by casp-6 in vitro. (A) Purified 5-LO protein (1 μg) was incubated with 0.3 units of either isolated recombinant casp-6 or casp-8 as described. After 16 h, aliquots were analyzed for full-length (78 kDa) and cleaved (58 kDa) 5-LO protein by WB. (B) Purified 5-LO protein (1 μg) was incubated with the indicated amounts of casp-6. After 16 h, aliquots were analyzed for 5-LO protein by WB. (C) Relevant Asp residues within 5-LO were replaced by Ala, and the proteins (1 μg) were expressed and purified as described and incubated with 0.3 units of casp-6. After 16 h, aliquots were analyzed for 5-LO protein by WB. The results are representative of at least three experiments.

Fig. 7.

Homology-based model of the 3D structure of 5-LO. (A) Model of 5-LO constructed from an x-ray structure of 15-LO (Protein Data Bank ID code 1lox). (B) Close-up model of the putative casp-6 cleavage site region in 5-LO, viewed along the direction of the arrow in A. The scissile bond is between Asp-170 and Ser-171. The casp-6 recognition sequence IQFD is supposed to form a loop-like structure that is connected to the C2-like domain via a single hydrogen bond between Asp-166 and the backbone amide of Trp-102.